Biomedical subjects

J G Levy

Publications and source records attributed to J G Levy.

At least 73 records · Page 4Linked to original sources

PubMed1991

Photodynamic killing of human squamous cell carcinoma cells using a monoclonal antibody-photosensitizer conjugate.

We have developed procedures in which the photosensitizer benzoporphyrin derivative monoacid ring A (BPD) can be covalently linked to carrier molecules of modified polyvinyl alcohol (PVA) to produce water-soluble PVA-BPD conjugates with a molecular mass in the range of 30 kd. These carriers can subsequently be covalently linked to monoclonal antibodies (MoAbs) using heterobifunctional linking agents. We describe here such a conjugate in which the MoAb (5E8) has specificity for a glycoprotein detected on human squamous cell carcinomas of the lung. We provide evidence that the conjugates produced were covalently linked and retained both their photosensitizing and antigen-binding activities. We show further that the MoAb-PVA-BPD conjugate, in the presence of 10% fetal calf serum, exhibited highly enhanced phototoxic killing of the target cell line (A549) over that exhibited by free BPD or a control MoAb-PVA-BPD conjugate. These results demonstrate, therefore, both the selectivity and specificity of this MoAb conjugate.

Antibodies, Monoclonal↗

PubMed1991

Photosensitising potency of structural analogues of benzoporphyrin derivative (BPD) in a mouse tumour model.

The in vivo characteristics of four analogues of benzoporphyrin derivative (BPD) have been investigated. Biodistribution data obtained in DBA/2J mice with BPD-MA (monoacid ring A analogue) which had been tritiated or internally labelled with 14C showed that both labelled materials acted in an essentially identical manner during the period of study. Biodistribution and clearance studies showed that relative distribution in a variety of mouse tissues was similar for all BPD analogues. M1 tumour cells (rhabdomyosarcoma in DBA/2J mice) taken from tumours excised from animals treated 3 h earlier with BPD, and tested in vitro for photosensitivity provided evidence that significant levels of photosensitiser detected in tumour was both active and associated with tumour cells. The monoacid forms of BPD were found to be much more photodynamically active in this test than were the diacid analogues. The ability of the analogues to ablate tumours in mice by photodynamic therapy was also tested. Again, BPD-MA and BPD-MB proved to be measurably better than the diacid analogues. These findings are discussed in reference to structural and physical differences between the analogues.

Animals↗

PubMed1991

The effects of plasma lipoproteins on in vitro tumor cell killing and in vivo tumor photosensitization with benzoporphyrin derivative.

The influence of lipoprotein association on in vitro tumor cell killing and in vivo tumor photosensitization with benzoporphyrin derivative (BPD) has been investigated in M-1 tumor bearing mice. The association of benzoporphyrin mono acid ring A with either low or high density lipoprotein increased tumor cell killing in an in vivo/in vitro cytotoxicity assay performed 3 h post intravenous drug administration. Eight hours following photosensitizer injection only low density lipoprotein (LDL) mixtures produced significant (P less than or equal to 0.005) increases in tumor cell killing compared to BPD in unfractionated plasma. The efficacy of in vivo photosensitization in the presence of lipoproteins correlated with the in vivo/in vitro cytotoxicity. Association of BPD with low or high density lipoproteins resulted in delayed tumor regrowth and higher cure rates when light exposure (125J/cm2) was performed 3 h post drug administration. When light exposure was performed 8 h post-injection only LDL-BPD mixtures led to enhanced tumor eradication compared to BPD administered in aqueous solution or unfractionated plasma.

Animals↗

PubMed1991

Mouse skin photosensitization with benzoporphyrin derivatives and Photofrin: macroscopic and microscopic evaluation.

A comparative study, at both the macroscopic and microscopic level, of skin photosensitivity caused by four isomeric forms of benzoporphyrin derivative (BPD) has been carried out, and compared to effects of Photofrin. Animals injected intravenously with BPD analogues and exposed to light 3 h later showed extensive photosensitivity. Animals receiving the monoacid derivatives of BPD (BPD-MA and BPD-MB) showed markedly more photosensitivity than those receiving the diacid derivatives (BPD-DA and BPD-DB). Animals receiving BPD analogues which were exposed to light 24 h or more later showed only minimal reactivity. Histological examination of biopsies taken after photosensitizer injection and light exposure showed extensive changes in epidermis and dermis, including epidermal erosion, degranulation of the stratum granulosum, spongiosis, depletion in cellularity and mast cell degranulation. These changes were noted to be similar to changes caused by Photofrin.

Animals↗

PubMed1991

Purification of an in vitro inhibitor of normal myelopoiesis using a monoclonal antibody directed to a common antigen of myelogenous leukemia (CAMAL).

A monoclonal antibody, CAMAL-1, has been previously shown to react specifically and at high frequencies with cells of patients with acute myelogenous leukemia (AML) and chronic granulocytic leukemia (CGL). High expression of this antigen in remission patients' bone marrow cells was shown to correlate with both relapse and lower survival times. Preliminary studies showed that material extracted from leukemic cells and eluted from CAMAL-1 immunoadsorbent columns profoundly inhibited the formation of normal colony-forming units (CFU) but had no effect on formation of such colonies from cells of patients with CGL. We have used CAMAL-1 affinity chromatography in combination with FPLC gel filtration to purify the inhibitory material from leukemic cell extracts. We have successfully isolated a 30-kd component (P30) that functions as an inhibitor of normal myelopoiesis in vitro; when P30 was added to normal progenitor cell assays it significantly inhibited the growth of normal CFU but had no inhibitory effect on the growth of CGL progenitor cells at equivalent concentrations. The inhibitory effect is preferentially directed to granulocytic progenitors. Antibodies raised to P30 reacted in Western blot analyses with affinity purified material from patients' cells as well as with a 30-kd component in the cell lysates and supernatants of the leukemic cell lines HL60 and K562; this finding suggested that the P30 inhibitory component might be produced by leukemic cells.

Amino Acid Sequence↗

PubMed1990

Development of technology for linking photosensitizers to a model monoclonal antibody.

A procedure is described whereby the photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA) was covalently linked to a model monoclonal antibody in a manner which is reproducible, quantifiable, and retains both the biological activity of the antibody and the cytotoxicity of the photosensitizer. Preliminary steps involved the linkage of BPD-MA to a modified polyvinyl alcohol (PVA) backbone, followed by conjugation to the antibody using heterobifunctional linking technology. Briefly, polyvinyl alcohol (MW ca. 10,000) was modified with 2-fluoro-1-methyl pyridinium toluene-4-sulfonate and 1,6-hexanediamine to produce side chains containing free amino groups. The free carboxyl group of BPD-MA was utilized to conjugate photosensitizer molecules to modified PVA using a standard carbodiimide reaction. Final linkage of the PVA-BPD to a model monoclonal antibody involved further substitution of the carrier with 3-mercaptopropionic acid and carbodiimide to introduce 3-4 sulfhydryl residues per carrier molecule, and introduction of sulfo-m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester residues to the monoclonal (3-4 residues/molecule). Conjugation was effected by reaction of the two species at pH 5.5 for 18 h. Detailed methodology and tests for efficacy of the procedure are provided.

Antibodies, Monoclonal↗

PubMed1990

Biodistribution of tritiated benzoporphyrin derivative (3H-BPD-MA), a new potent photosensitizer, in normal and tumor-bearing mice.

The biodistribution of a new and very potent photosensitizer, benzoporphyrin derivative-monoacid, ring A (BPD-MA), was determined in normal and P815 (mastocytoma) or M1 (rhabdomyosarcoma) tumor-bearing DBA/2J mice. A dose of 80 micrograms of 3H-BPD-MA was determined at 3, 24, 48, 72, 96 and 168 h post injection. The following tissues were tested: blood, brain, heart, intestine, kidney, lung, liver, muscle, skin, stomach, spleen, thymus and tumor. The biodistribution of 3H-BPD-MA in normal and tumor-bearing mice was comparable overall. 3H-BPD-MA localized in tumors better than in other tissues except kidney, liver and spleen. The tumor to tissue ratios were in the range 1.5-3 at 24 h post injection and increased further during the next 72 h. The highest levels of 3H-BPD-MA were observed in all tissues at 3 h post injection and decreased rapidly during the first 24 h. After 24 h the clearance from tissues was rather slow. The preliminary clearance data obtained in a group of five normal mice indicated that the majority of the injected dose (60%) cleared from the body via the bile and feces, while only about 4% cleared via kidneys and urine. Studies in which 3H-BPD-MA was extracted from tumor, kidney and liver 3 and 24 h after injection showed that, at 3 h, all the photosensitizing activity in tumor was retained. At 24 h only 39% of the activity was retained and considerably less active material was present in liver and kidney.

Animals↗

PubMed1990

The role of suppressor factors in the regulation of immune responses by ultraviolet radiation-induced suppressor T lymphocytes. III. Isolation of a suppressor factor with the B16G monoclonal antibody.

The purpose of this study was to determine whether the ultraviolet (UV) radiation-induced systemic suppression of the immune response results from the release of soluble suppressor factors (TsF) by UV-induced suppressor T cells (UV Ts). Injecting a TsF-specific monoclonal antibody (B16G) significantly reduced the UV radiation-induced suppression of contact hypersensitivity (CHS). The transfer of spleen cells from the UV-irradiated, B16G-treated mice into normal recipients suppressed CHS in the recipients, indicating that while the suppression of CHS was reversed in the UV-irradiated, B16G-treated mice, suppressor cells were still present. Supernatants from cultures containing UV Ts were incubated on B16G-immunoadsorbent columns. The antibody-bound fraction (45- to 60-kDa, non-disulfide-linked proteins) suppressed CHS when injected into normal recipients. These results demonstrate that the B16G antibody reacts with TsF from UV Ts and suggest that B16G acts in vivo by inhibiting the activity of TsF. Thus, suppressor factors appear to play an essential role in the regulation of immune responses by UV Ts.

Animals↗

PubMed1990

Preferential uptake of benzoporphyrin derivative by leukemic versus normal cells.

Benzoporphyrin derivatives (BPDs) are photosensitizers, which fluoresce strongly at 690 nm, and may be candidates for various applications of photodynamic therapy (PDT). Fluorescence-activated cell sorting (FACS) analysis, subsequent to ultraviolet light excitation, revealed pronounced differences in red fluorescence between leukemic cell lines (HL60, K562 and L1210), leukemic clinical isolates, and normal human or murine bone marrow cells incubated with BPD. These observed differences in BPD-mediated fluorescence provide the rationale for sorting leukemic from normal cells via FACS or may constitute a novel method for extracorporeal purging of remission marrow by photodynamic therapy in autologous bone marrow transplantation.

Animals↗

PubMed1990

Tumour scanning with indium-111 dihaematoporphyrin ether.

Photofrin II (dihaematoporphyrin ether/ester, DHE) was labelled with indium-111 and its biodistribution in tumour bearing mice compared with that of 111In chloride. The uptake and clearance of 111In labelled DHE differed markedly from that of indium-111 chloride in that the former was not taken up by the tissues as much as the latter. Scintillation scanning with a gamma-camera showed marked uptake of both 111In agents at the site of the tumour, but a much lower tissue background (excluding the abdominal organs) for the mice given 111In DHE. Tumour:muscle ratios of dissected tissues were 2-3 times higher in 111In DHE treated animals as compared to the uptake of 111In chloride. There was a distinct difference in the pattern of distribution of the two 111In preparations in the tissues. The major accumulation of 111In chloride was in the kidneys, whereas the highest uptake of 111In DHE was in the liver, the organ in which unlabelled porphyrins accumulate. Extraction and testing of materials from tumours of 111In DHE treated animals indicated that most of the tumour extractable 111In had remained associated with the porphyrin in vivo up to 4 days after injection.

Animals↗

PubMed1990

In vitro evaluation of phototoxic properties of four structurally related benzoporphyrin derivatives.

Four structural analogs of benzoporphyrin derivative (BPD) have been studied and compared for photosensitizing activity in vitro. All analogs have an identical reduced tetrapyrrol porphyrin ring, and differ by the position of a cyclohexadiene ring (fused at either ring A or ring B of the porphyrin) and the presence of either two acid groups or one acid and one ester group at rings C and D of the porphyrin. Photosensitizer activity was tested with the M1 tumor cell line using an assay (the MTT assay) which detects mitochondrial hydrogenases as a measure of cell viability. This assay was shown to be equivalent to the standard clonogenicity or [3H]thymidine uptake assay. Comparative studies with the BPD analogs showed that the monoacid derivatives had equivalent cytotoxicity and were about five-fold more active than the diacid forms. This was the case whether the assays were performed in the presence or absence of fetal calf serum.

Animals↗

PubMed1990

The plasma distribution of benzoporphyrin derivative and the effects of plasma lipoproteins on its biodistribution.

The plasma distribution and biodistribution of benzoporphyrin derivative were examined. Two analogs of benzoporphyrin derivative were mixed with human plasma in vitro and recovered in the lipoprotein fractions upon separation by chromatography or ultracentrifugation. The majority of both analogs was recovered with high density lipoprotein. The effect of prebinding benzoporphyrin derivative to lipoproteins on the biodistribution of the drug in vivo was studied in tumor bearing DBA/2J mice. At 3, 8 and 24 h post-injection, tumor and tissue samples were excised and analyzed for benzoporphyrin derivative content. Precomplexing benzoporphyrin derivative with low density lipoprotein or high density lipoprotein led to significantly (P less than 0.05) greater tumor accumulation than in aqueous solution.

Animals↗

PubMed1989

Preadministration of a T-suppressor factor enhances tumor immunity in DBA/2 mice.

Previously we have described the isolation and characterization of a T-suppressor factor (TsF) from a T cell hybridoma (A10F), which has a degree of specificity for the DBA/2 mastocytoma P815. Administration of A10F intravenously at the time of tumor cell injection resulted in an accelerated rate of tumor growth, decreased cytotoxic T lymphocyte antitumor activity, and reduced survival time. In the work reported here, we have shown that administration of affinity-enriched A10F 7-14 days prior to tumor cell injection causes what appear to be reverse effects, in that an enhanced resistance to the P815 tumor is observed in vivo, an effect which we can correlate with the demonstration of antitumor cytotoxic T lymphocyte activity in vitro. These effects are dose-dependent since only doses of TsF at 20 micrograms or greater are effective. A similar effect was found when A10F was administered to DBA/2 mice 10 days prior to challenge with two unrelated tumors (L1210 and M-1). However, when another TsF (Fd11F) with apparent specificity for a nominal antigen was tested in this system, it had no effect on tumor growth.

Adjuvants, Immunologic↗

PubMed1989

Isolation and characterization of a T-suppressor factor specific for the lupus-associated autoantigen RNP-Sm.

We have previously described a monoclonal antibody, B16G, which has been found to be specific for T-cell derived suppressor factors (TsF). B16G has been shown to react with T-suppressor cells, TsF in the spleen of normal or tumor-bearing mice, the TsF produced by tumour-specific, or hapten-specific T-cell hybridomas, and with polyclonal whole human TsF isolated from tonsillar tissue. This pan-reactivity inherent to the B16G antibody suggests that it recognizes some common, shared epitope of the TsF molecule. In this study, we have used B16G as a probe to isolate a TsF-producing T-cell hybridoma, S-50, from CBA mice (H-2k) that is specific for the Lupus-associated antigen, RNP-Sm. The TsF bound specifically to RNP-Sm and inhibited the production of anti-RNP-Sm antibody cell cultures from MRL-lpr mice. SDS-PAGE analysis of purified S-50 TsF revealed a B16G-reactive band with a molecular weight of 43 kd.

Animals↗

PubMed1989

CAMAL: evidence for an inhibitory role in normal granulopoiesis.

Maintenance of normal granulopoiesis requires complex interactions between positive (stimulatory) and negative (inhibitory) regulatory molecules, the cells that produce them and their targets. In myeloid leukemia these signals continue to operate but in an obviously unbalanced fashion, allowing the emergence and eventual dominance of the malignant clone. In this study, a common antigen in myeloid leukemia (CAMAL) has been shown to bind to normal leukocyte membranes. At similar concentrations (between 10 and 15 micrograms/ml), leukemia-derived CAMAL caused profound inhibition of normal colony growth in the myeloid progenitor cell assay but had no inhibitory effect on granulocyte-macrophage colony-forming unit (CFU-GM) growth from myeloid leukemia patients in active disease states. These preliminary results indicated the myeloid leukemia cells possessed apparent differences in responsiveness to CAMAL-mediated regulation compared to normal cells. Lack of or decreased responsiveness to inhibition by leukemia-derived CAMAL may facilitate dominance of the malignant clone over normal cells.

Antigens, Differentiation, Myelomonocytic↗

PubMed1988

Suppressor cell-inducing factor: a new lymphokine secreted by a natural suppressor cell line with natural cytotoxic activity. I. Purification to apparent homogeneity and initial characterization of suppressor cell-inducing factor.

The lymphokine suppressor cell-inducing factor (SIF), obtained from 15 liters of serum-free culture supernatants of the natural suppressor cell line, M1-A5, has been purified to apparent homogeneity by a combination of gel filtration, ion exchange chromatography, and reverse-phase-HPLC. Purity of SIF was assessed by the migration of the factor as a single band on SDS-PAGE, and the elution from reverse-phase-HPLC column as a single and sharp peak. SIF activity was retained after both procedures. Two protein factors with SIF activity were isolated from M1-A5 culture supernatants. The first protein factor (SIF alpha) had a Mr of 43 kDa, and the second protein factor (SIF beta) had a Mr of 6 kDa. Final purification of SIF alpha yielded 5 micrograms protein with specific activity of 4 x 10(6) U/mg protein. Final purification of SIF beta yielded 40 micrograms protein with specific activity of 7.5 x 10(7) U/mg protein. The relationship between SIF alpha and SIF beta, as well as the relationship with other suppressor factors, will be addressed.

Amino Acids↗

PubMed1988

Identification of a T cell subset using a rabbit antibody raised against a T suppressor molecule.

Rabbit antiserum to a unique component of an Ag-binding Ts-factor was generated by repeated immunization with purified 30-kDa protein isolated from Fd11 Ts factor (11). This antiserum (anti-p30) was shown to recognize cell surface determinants expressed on the Ts hybridomas Fd11 and A10 but not the fusion partner BW5147. Furthermore, this antiserum was shown to bind to approximately 4% of thymocytes and 10% of nylon wool-purified splenic T cells from all strains of mice tested. Sorting nylon wool-purified T cells from DBA/2 mice for the CD4+ and CD8+ subsets revealed both populations contained cells that bound anti-p30. In addition, when CD4-8- thymocytes were examined for anti-p30 labeling, it was found that about 30% of this enriched population also expressed p30 molecules. In a functional study, anti-p30 was able to neutralize the suppressive effects of Fd11 on a specific assay for in vitro antibody synthesis against ferredoxin.

Animals↗

PubMed1988

Suppressor deletion therapy: selective elimination of T suppressor cells in vivo using a hematoporphyrin conjugated monoclonal antibody permits animals to reject syngeneic tumor cells.

A MAb (B16G) which recognizes a constant epitope on TsC and their soluble factors in DBA/2 mice has been described previously. In this study, we show that when this MAb is covalently linked to the photoactivable molecule Hp, and injected i.v. into P815 tumor-bearing mice which were subsequently exposed to light, tumors undergo permanent regression in 10%-40% of these mice (depending on the individual experiment). All control animals died within an average of 22-24 days after tumor cell injection. It is suggested that tumor regression is attributable to immune mechanisms facilitated by the elimination of a population of TsC. When splenocytes of B16G-Hp-treated mice were assayed in vitro for the generation of CTL active against P815 tumor cells, it was found that 24 h after treatment, a significant increase in killer cell activity was noted but that this effect was gone by 48h. We also show that B16G-Hp conjugates are capable in vitro of specifically killing cells of a TsC hybridoma, A10 (which has been shown previously to secrete a T suppressor factor reactive with P815 cell surface antigens). This conjugate had no cytotoxic effect on P815 cells under conditions in which A10 cells were killed.

Animals↗